A novel concept has recently emerged demonstrating that monocytes, following infiltration into arteries, can differentiate into two major classical (M) or alternative (M2) macrophages during the development of atherosclerosis (ATH). In vitro preliminary data indicates that BMP-7 stimulated monocytes (THP-1 cells) differentiate into M2 polarized macrophages, which was confirmed with increased M2-specific marker CD206 and up regulation of released IL-10. In vivo preliminary data suggests that intravenous injection of BMP-7 in ApoE KO mice experiencing a partial ligated coronary artery (PLCA) have increased M2 polarized macrophages, increased BMP-7R on monocytes and M2 polarized macrophages, increased M2 macrophage- specific released IL-10 and IL-1 receptor antagonist, and increased blood velocity compared with sham controls at two weeks follow up. Accordingly, we propose that monocytes differentiate into M2 macrophages, an anti-inflammatory phenotype, under the influence of BMP-7 at different stages of ATH development. Additionally, we propose to understand whether M-CSF action is also required along with BMP-7 on monocyte differentiation to M2 polarized macrophages and to correlate the presence and influence of BMP-7 on M1 and M2 macrophages in ATH using heterozygous mice obtained from backcrossing op mice (M-CSF deficient) and ApoE deficient mice. We also propose to establish the mechanisms by which BMP-7 would affect macrophage polarization. Specific aims are as follows: Aim1a: To examine the presence of BMP-7 in the lesions during the development of ATH in ApoE KO animals and to correlate its presence with M1 and M2 types of macrophages. We will determine this using immunohistochemical and real-time PCR techniques and measuring pro-and anti- inflammatory cytokine levels, specific expression of inflammatory genes, and specific genes for M1 and M2 phenotype macrophages such as arginase and scavenger receptors. Furthermore, the presence and preponderance of specific macrophage phenotypes will be identified. Aim 1b: We will use loss of function of BMP-7R using siRNA methodology and inhibitors for BMP-7 to determine macrophage polarization. Aim 2: To determine if BMP-7 induced monocytes to M2 polarization is mediated via BMP-7 alone or if influence is provided by released M-CSF from the monocytes. We will establish whether a) BMP-7 induces differentiation of monocytes into M2 macrophages, or whether b) BMP-7 and M-CSF both induce monocytes into M2 polarized macrophages. Genetically altered op mice specific for M-CSF gene deletion will be backcrossed with the ApoE KO mice, heterozygous mice will be subjected to PLCA surgery, and all parameters will be established as proposed in Aim 1. Aim 3: We will determine SMAD-dependent and p38, JNK, and ERK pathways and their correlation with the release/expression of specific phenotype-associated gene expressions. Results of these studies will define the impact of BMP-7 on M2 polarized macrophages, anti-inflammatory cytokines, their effects on gene expression, and molecular mechanisms in the progression of ATH.